Xylitol is a naturally occurring 5-carbon sugar alcohol, which is present in small amount in many fruits and vegetables and is produced in the human body during normal metabolism. It has approximately the same sweetness as sucrose, lower caloric content of about 2.4 kcal/g, and it has certain known metabolic, dental and technical characteristics which make it an attractive special sweetener or sugar substitute in various contexts. For instance, xylitol is cariostatic and even anti-cariogenic. It is metabolised independently of insulin and can be safely consumed by non-insulin dependent diabetics, and it is non-toxic. Nowadays it is widely used in chewing gums, dental care products, health promoting products, functional food products, pharmaceutical products, confectionery products and the like.
Xylitol is usually prepared by processes utilizing natural raw materials, especially xylan-containing materials. In current use are methods in which a xylan-containing material is first hydrolysed to produce a mixture of monosaccharides, including D-xylose. After purification the D-xylose is then converted to xylitol, generally in a chemical process using e.g. a nickel catalyst such as Raney-nickel. A number of processes of this type have been described in the literature of the art. U.S. Pat. No. 3,784,408 (Jaffe et al.), U.S. Pat. No. 4,066,711 (Melaja et al.), U.S. Pat. No. 4,075,406 (Melaja et al.), U.S. Pat. No. 4,008,285 (Melaja et al.) and U.S. Pat. No. 3,586,537 (Steiner et al.) may be mentioned as examples.
The recovery of D-xylose during wood and pulp processing can be performed by various separation techniques. Chromatography is widely used. A process for the fractionation of sulfite spent liquor by chromatography is described in U.S. Pat. No. 5,737,225, Xyrofin Oy. The process uses a simulated moving bed including at least two chromatographic beds and, preferably, at least three different fractions are recovered, one of these being enriched with xylose and another with lignosulphonates. For instance U.S. Pat. Nos. 4,631,129; 4,008,285 and 4,075,406 also describe chromatographic processes for the recovery of xylose.
Processes in which microorganisms are utilised for biotechnological production of xylitol have also been described. It is known that many yeast strains produce reductase enzymes that catalyse the reduction of sugars to corresponding sugar alcohols. Many yeasts, in particular Pichia, Candida, Hansenula and Kluyveromyces, are also capable of reducing xylose to xylitol as an initial step in their xylose metabolism.
The reaction route or pathway of xylose utilisation for yeasts is in general the following: xylitol is synthesised in the first step by reduction of xylose to xylitol with the aid of xylose reductase. Xylitol is then metabolised by a series of successive steps. Xylitol is first oxidised to xylulose with xylitol dehydrogenase, xylulose is phosphorylated to xylulose-5-phosphate with xylulose kinase (also called xylulokinase), and then part of the xylulose-5-phosphate is converted to pyruvate via several intermediate steps. Also ethanol and CO2 can be formed. The reactions are not tightly coupled, and the relevant main products and by-products vary depending on the yeast strain and the fermentation conditions, such as oxygen availability.
For instance PCT publications WO 90/8193, WO 91/0740, WO 88/5467 and French published application 2 641 545 describe the use of Candida tropicalis, Candida guilliermondii and Candida parapsilosis, respectively, for the industrial production of xylitol.
U.S. Pat. No. 5,081,026, Heikkilä et al., describes a process for the production of xylitol from xylose, in which an aqueous xylose solution is fermented with a yeast strain capable of converting free xylose to xylitol and free hexoses to ethanol. After fermentation, a xylitol-rich fraction is obtained by chromatographic separation, and finally, xylitol is recovered from said fraction.
Genetic modification of microorganisms in order to enhance their xylitol production have also been reported in the literature of the art. For example, in WO 91/15588, Hallborn, J. et al. describe the cloning of the xylose reductase gene from Pichia stipitis into Saccharomyces cerevisiae. Gong C. et al., Biotechnol. Letters 3:125-130 (1981) describe two high xylitol producing yeast mutants denominated HXP1 and HXP2, obtained after UV-mutagenesis of a wild strain of Candida tropicalis which originally was capable of metabolising D-xylose into xylitol.
EP 0 604 429, Xyrofin, describes novel yeast strains with modified xylitol metabolism, a process for the production of said strains, and the use of said strains in a process for producing xylitol. The strains are capable of reducing xylose into xylitol, but are deficient in one or more enzymes involved in the xylitol metabolism, with the effect that the xylitol produced accumulates in the culture medium and can be recovered therefrom. The yeasts described belong to the genera Candida, Hansenula, Kluyveromyces or Pichia, and the genetic modification eliminates or reduces expression of the gene that encodes xylitol dehydrogenase or xylulose kinase, or both.
Another approach that has been suggested for the bioproduction of xylitol is the enhancement of xylose production, thus providing more xylose as the primary metabolite for xylitol production.
Some fungi, including Aureobasidium, Aspergillus, Trichoderma, Fusarium and Penicillium, have been reported to have xylanolytic activity and thus be able to degrade xylan-containing biopolymers into xylose. E.g. Kuhad R. C. et al., Process Biochemistry 33:641-647 (1998) describe a hyperxylanolytic mutant strain of Fusarium oxysporum produced by UV and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment.
EP 0 672 161, Xyrofin, describes a method for the production of xylitol from carbon sources other than xylose and xylulose by using recombinant hosts. The microorganisms produce xylitol via an altered arabitol route involving in particular arabitol dehydrogenase, and/or via altered (over)expression of genes encoding the enzymes of the oxidative branch of the pentose phosphate pathway (PPP), in particular glucose-6-phosphate dehydrogenase or 6-phospho-D-gluconate dehydrogenase, thus enabling utilisation of glucose, for instance. When used, D-glucose is phosphorylated into D-glucose-6-phosphate and converted to D-ribulose-5-phosphate via 6-phospho-D-gluconate. The D-ribulose-5-phosphate is then epimerised to D-xylulose-5-phosphate, dephosphorylated to D-xylulose and reduced to xylitol. Amplification of glucose-6-phosphate dehydrogenase enzyme activity in osmotolerant yeasts is later also described in FR 2 772 788, Roquette Freres.
U.S. Pat. No. 5,096,820, Leleu et al., also describes a process in which xylitol is produced from D-glucose. Glucose is first microbiologically converted to D-arabitol, which likewise is microbiologically converted to D-xylulose. The D-xylulose is then enzymatically isomerised into a mixture of D-xylose and D-xylulose, which is catalytically hydrogenated. Finally, the xylitol is recovered by chromatographic separation or crystallisation. The D-arabitol containing fractions, or the mother liquor from crystallization, which are rich in xylitol but also in D-arabitol, are preferably recirculated into the process. U.S. Pat. No. 5,238,826, Leleu et al., uses a similar process to obtain D-xylose, ultimately for the preparation of xylitol by hydrogenation. Also in this process, D-glucose is first microbiologically converted to D-arabitol, which then likewise is microbiologically converted to D-xylulose. The D-xylulose is then enzymatically isomerised into a mixture of D-xylose and D-xylulose. Finally, the mixture is subjected to chromatographic separation, the D-xylose fraction is recovered and the D-xylulose fraction is recirculated into the isomerisation step.
The background art thus describes the production of xylitol from naturally occurring raw materials. At present, the raw materials mainly used are xylan-containing materials. From xylan, xylitol is produced by chemical processes or combinations of chemical and biological processes. Further, processes utilising microorganisms, in particular yeasts, capable of producing xylitol from monosaccharide solutions or pure D-xylose solutions have been described.
In view of the increasing use of xylitol, in particular due to its properties as sweetener and therapeutic effects, new methods for the production of xylitol would be welcome. In particular, there is an expressed need for processes for the production of xylitol from other sources than those mainly utilised.
U.S. Pat. No. 5,714,602, Cerestar Holding B.V., discloses a process developed from the this viewpoint. According to the document, xylitol is produced from gluconic acid. In a first step, gluconic acid is decarboxylated, by using sodiumhypochlorite or hydrogen peroxide into arabinose, which through hydrogenation is converted into arabinitol. After epimerisation, a mixture of xylitol, ribitol and D,L-arabinitol is obtained, from which xylitol is recovered by chromatographic methods.
EP 754 758, Cerestar Holding B.V., relates to a process for the production of xylitol in two steps. In the first step a hexose is converted to a pentitol by fermentation, and in the second step the pentitol is catalytically isomerised to yield a pentitol mixture. Specifically, the document describes a process in which glucose is fermented to arabinitol and then isomerised into a pentitol mixture containing xylitol, ribitol and D,L-arabinitol. Xylitol can be recovered from said mixture by chromatographic methods.
WO 93/1903, Amylum, also describes a process for the production of xylitol from monosaccharides, in particular D-glucose, D-fructose, D-galactose, L-sorbose or mixtures thereof. The starting material is first oxidized to D-arabinonic acid, D-lyxonic acid, and/or L-xylonic acid and the intermediate is then hydrogenated in one or several steps to a product consisting mainly of xylitol or a mixture of xylitol, arabinitol and ribitol. Finally, if necessary, xylitol is separated by means of chromatography.